Pregnancy detection

ABSTRACT

A METHOD OF IN VITRO PREGNANCY DETECTION COMPRISES CONTACTING A URINE SAMPLE OF A FEMALE WITH A DIAGNOSTIC COMPOSITION CONSISTING ESSENTIALLY OF BROMOCRESOL PURPLE OR CHLOROPHENOL RED AND OBSERVING THE COLOR PRODUCED.

United States Patent 3,595,620 PREGNANCY DETECTION Harry W. Gordon,Bronx, N.Y., assignor to Julius Schmid, Inc., New York, N.Y. N0 Drawing.Filed Oct. 28, 1968, Ser. No. 771,363 Int. Cl. G01n 21/06, 31/22, 33/16US. Cl. 23230 8 Claims ABSTRACT OF THE DISCLOSURE A method of in vitropregnancy detection comprises contacting a urine sample of a female witha diagnostic composition consisting essentially of Bromocresol purple orChlorophenol Red and observing the color produced.

This invention relates to a diagnostic dye indicator for in vitropregnancy detection.

It is known that human chorionic gonadotrophin (hereinafter referred toas HCG) is produced during pregnancy by the placenta and is foundshortly after nidation in serum and urine. HCG is secreted in the urineof pregnant women and is not found in the urine of non-pregnant womenexcept non-pregnant women afflicted with certain pathologicalconditions. A substantial number of the prior art pregnancy tests arebased on in vivo and in vitro methods of determining the presence ofHCG. Most of these tests have numerous disadvantages or drawbacks amongwhich are: inordinate length of time required for the test, complicatedtest procedures requiring careful following of instructions, complicatedprocedures required for preparing the diagnostic reagent, lack ofsensitivity and specificity of the tests and lack of stability of thereagents. Moreover, some of the in vitro tests known in the art requirethe maintenance of a large supply of animals of the required age and theneed for multiple injections.

Accordingly, there has now been discovered a quick and simple procedurefor in vitro pregnancy detection based on the use of an indicator dyewhich overcomes numerous disadvantages associated with the prior artpregnancy test procedures.

Thus, one aspect of the present invention is to provide an in vitromethod of determining pregnancy in women and other female mammals whichdoes not require any laboratory equipment or any special skills forinterpretation of the results and which can be routinely carried out bya layman.

Another aspect of the present invention is to provide in vitrodiagnostic reagents which are characterized by a high degree ofsensitivity and provide accurate results in substantially faster timethan heretofore possible.

An additional aspect of the present invention is to provide a diagnosticdye reagent for determining pregnancy which consists essentially of abuffered Bromocresol purple solution having a pH between about 5.5 and7.

A further aspect of the present invention relates to a process ofproducing diagnostic dye reagents of this invention.

These and other aspects of the present invention will become apparentfrom the following description.

In accordance with my invention, it has been discovered that certaindyes having a pH between about 5.5 and 7 and preferably between about 6and 6.5 will act as an indicator when added to female urine therebyproviding a simple and accurate test for pregnancy detection. Thepreferred dye used as a diagnostic reagent is Bromocresol purple (',5"dibromo-o-cresolsulfonephthalein) solution containing a buffering agent.However, a closely related dye, namely Chlorophenol Red (3',3"dichlorophenolsulfonephthalein) can also be used. When the 3,595,620Patented July 27, 1971 ice Bromocresol purple solution is added to asample of female urine it has been found to turn a deep purple in theurine of a female who is pregnant and remain red to reddish-brown in aurine sample of a patient not pregnant. If the pH of the dye is belowabout 5.5 and above about 7 the diagnostic reagent does not providesatisfactory results.

A preferred procedure for preparing the dye reagent is to include abuffering agent in the solution to increase the pH stability of thesolution during extended periods of storage. The selection of thebuffering agent is not critical. Suitable materials include phosphates,glycine, borates, etc.

A Bromocresol buffered solution used in my invention may be prepared asfollows: To 0.40 gram of Bromocresol purple in a glass mortar, there wasadded a .01 N sodium hydroxide solution in increments of about 10 ml.while vigorously stirring, until a total of 74 ml. has been added.Stirring is continued until solution is completed. Thereafter about 725ml. of a phosphate buffer (pH 6) solution (250 ml. 0.2 M potassiumphosphate monobasic solution mixed with 28 ml. 0.2 M sodium hydroxidesolution and diluted to 1000 ml. with water) was thoroughly mixed withthe Bromocresol dissolved in the sodium hydroxide to provide a bufferedsolution having a pH of about 6.

The in vitro diagnostic dye reagent may also be prepared by grinding 0.1gram of Bromocresol purple in a mortar with 18.5 ccs. of 0.01 N sodiumhydroxide to produce a homogenous mixture. Thereafter this mixture isdiluted to 250 ccs. with distilled water to provide a red solutionhaving a pH of about 6. It has been found that if the dye is addeddirectly to the water and the sodium hydroxide separately added, theresulting dye solution will not produce a sufiiciently distinct colorchange when added to urine of a pregnant woman.

The procedure for adding the diagnostic dye reagent to the urine sampleis as follows: One to two drops of the diagnostic dye reagent are addedto 3 to 5 ccs. of urine in a test tube which is shaken. The colordevelopment which takes place almost instantaneously is then observed.-As previously indicated a purple color indicates a positive reactionand a red to reddish-brown color a negative reaction. The quantity ofdiagnostic dye reagent used is not critical. Any substantial variationin the quantity of dye added to a given quantity of urine affects onlythe intensity of the color observed.

The eflicacy of the diagnostic dye reagent of my invention has beendemonstrated experimentally on a substantial number of patients undermedical supervision. To date these tests have shown a reliability ofbetween and False positive readings have been observed in a fewinstances where the female is taking a certain type of steroidcontaining drug. The purple color observed with these false readings issubstantially lighter or fainter than the intense purple color producedin those cases where a positive reaction is observed in female urinecontaining HCG.

Hence, the diagnostic dye reagent may be useful as a tool for the invitro detection of steroids and therefore permit a female who is takingan oral contraceptive to readily determine whether she has missed takinga pill in the prescribed cycle.

While the invention has been described with reference to the specificembodiments, it is to be understood that it is not to be limitedthereto, but is to be construed broadly and restricted solely by thescope of the appended claims.

What is claimed is:

1. A method of in vitro pregnancy detection in a female which comprisesadding to urine derived from said female a sufiicient quantity of acomposition consisting essential- 1y of a Chlorophenol Red solutionhaving a pH between about 6 and about 6.5 and observing the colorproduced in said urine.

2. A method of in vitro pregnancy detection in a female which comprisesadding to urine derived from said female a sufficient quantity of acomposition consisting essentially of a Bromocresol purple solutionhaving a pH between about 6 and about 6.5 and observing the colorproduced in said urine.

3. A method of detecting pregnancy in a female which comprisescontacting urine derived from said female with a diagnostic compositionselected from the class consisting of Bromocresol purple andChlorophenol Red and observing the color produced, said diagnosticcomposition having a pH between about 5.5 and about 6.5.

4. A method according to claim 3 wherein said diagnostic compositioncontains a buffering agent.

5. A method according to claim 3 wherein said diagnostic composition isBromocresol purple.

6. A method according to claim 5 wherein the pH of said Bromocresolpurple diagnostic composition is about 6.

7. A method according to claim 6 wherein said diagnostic composition isin the form of an aqueous solution.

8. A method of in vitro pregnancy detection in a female which comprisesadding to urine derived from said female a sufiicient quantity of acomposition consisting essentially of Bromocresol purple and a phosphatebuffer, said composition having a pH of about 6, and observing the colorproduced in said urine.

References Cited UNITED STATES PATENTS 8/1962 Collins 23230B1O 1/1967Fossel 23230Bl0 OTHER REFERENCES MORRIS O. WOLK, Primary Examiner E. A.KATZ, Assistant Examiner US. Cl. X.R. 252408; 424-7

